Multivalent immunogenic composition containing RSV subunit composition and influenza virus preparation

ABSTRACT

Immunogenic compositions for administration to adults, particularly to the elderly, to protect them against disease caused by infection by respiratory syncytial virus and by influenza virus comprise an immunoeffective amount of a mixture of purified fusion (F) protein, attachment (G) protein and matrix (M) protein of RSV and an immunoeffective amount of a non-virulent influenza virus preparation. The components of the composition, when formulated as a vaccine for in vivo administration, do not impair the immunogenicity of each other. The immunogenic composition may also contain an adjuvant.

This application is a national phase application under 35 U.S.C. 371 ofPCT/CA99/01194 filed Dec. 16, 1999, which is a continuation-in-part ofU.S. patent application Ser. No. 09/213,770 filed Dec. 17, 1998.

FIELD OF INVENTION

This invention relates to multivalent immunogenic composition,particularly for administration to adults.

BACKGROUND TO THE INVENTION

Human respiratory syncytial virus is the main cause of lower respiratorytract infections among infants and young children (refs. 1 to 3—a listof references appears at the end of the disclosure and each of thereferences in the list is incorporated herein by reference thereto).Globally, 65 million infections occur every year resulting in 160,000deaths (ref. 4). In the USA alone, 100,000 children may requirehospitalization for pneumonia and bronchiolitis caused by RS virus in asingle year (refs. 5, 6). Providing inpatient and ambulatory care forchildren with RS virus infections costs in excess of $340 millionannually in the USA (ref. 7). Severe lower respiratory tract disease dueto RS virus infection predominantly occurs in infants two to six monthsof age (ref. 8). Approximately 4,000 infants in the USA die each yearfrom complications arising from severe respiratory tract disease causedby infection with RS virus and parainfluenza type 3 virus (PIV-3). TheWorld Health Organization (WHO) and the National Institute of Allergyand Infectious Disease (NIAID) vaccine advisory committees have rankedRS virus second only to HIV for vaccine development.

RSV infection in adults was initially considered a significant problemonly in certain high-risk populations, such as the institutionalizedelderly. However, evidence has been accumulating that the infectionoccurs frequently in previously healthy adults (ref. 9).

RSV infections in the elderly usually represent reinfections in thosewho have had many prior episodes. These infections have been reported tocause altered airway resistance and exacerbration of chronic obstructivelung disease.

In adults over 60 years old, RSV usually causes mild nasal congestionand may also result in fever, anorexia, pneumonia, brochitis and deaths(ref. 10).

The structure and composition of RSV has been elucidated and isdescribed in detail in the textbook “Fields Virology”, Fields, B. N. etal. Raven Press, N.Y. (1996), in particular, Chapter 44, pp 1313–1351“Respiratory Syncytial Virus” by Collins, P., McIntosh, K., and Chanock,R. M. (ref. 11).

The two major protective antigens of RSV are the envelope fusion (F) andattachment (G) glycoproteins (ref 12). The F protein is synthesized asan about 68 kDa precursor molecule (F₀) which is proteolytically cleavedinto disulfide-linked F₁ (about 48 kDa) and F₂ (about 20 kDa)polypeptide fragments (ref. 13). The G protein (about 33 kDa) is heavilyO-glycosylated giving rise to a glycoprotein of apparent molecularweight of about 90 kDa (ref. 14). Two broad subtypes of RS virus havebeen defined A and B (ref. 15). The major antigenic differences betweenthese subtypes are found in the G glycoprotein while the F glycoproteinis more conserved (refs. 7, 16).

In addition to the antibody response generated by the F and Gglycoproteins, human cytotoxic T cells produced by RSV infection havebeen shown to recognize the RSV F protein, matrix protein M,nucleoprotein N, small hydrophobic protein SH, and the nonstructuralprotein 1b (ref. 17).

A safe and effective RSV vaccine is not available and is urgentlyneeded. Approaches to the development of RS virus vaccines have includedinactivation of the virus with formalin (ref. 18), isolation ofcold-adapted and/or temperature-sensitive mutant viruses (ref. 19) andpurified F or G glycoproteins (refs. 20, 21, 22). Clinical trial resultshave shown that both live attenuated and formalin-inactivated vaccinesfailed to adequately protect vaccines against RS virus infection (refs.23 to 25). Problems encountered with attenuated cold-adapted and/ortemperature-sensitive RS virus mutants administered intranasallyincluded clinical morbidity, genetic instability and overattenuation(refs. 26 to 28). A live RS virus vaccine administered subcutaneouslyalso was not efficacious (ref. 29). Inactivated RS viral vaccines havetypically been prepared using formaldehyde as the inactivating agent.Murphy et al. (ref. 30) have reported data on the immune response ininfants and children immunized with formalin-inactivated RS virus.Infants (2 to 6 months of age) developed a high titre of antibodies tothe F glycoprotein but had a poor response to the G protein. Olderindividuals (7 to 40 months of age) developed titres of F and Gantibodies comparable to those in children who were infected with RSvirus. However, both infants and children developed a lower level ofneutralizing antibodies than did individuals of comparable age withnatural RS virus infections. The unbalanced immune response, with hightitres of antibodies to the main immunogenic RS virus proteins F(fusion) and G (attachment) proteins but a low neutralizing antibodytitre, may be in part due to alterations of important epitopes in the Fand G glycoproteins by the formalin treatment. Furthermore, some infantswho received the formalin-inactivated RS virus vaccine developed a moreserious lower respiratory tract disease following subsequent exposure tonatural RS virus than did non-immunized individuals (refs. 24, 25). Theformalin-inactivated RS virus vaccines, therefore, have been deemedunacceptable for human use.

Evidence of an aberrant immune response also was seen in cotton ratsimmunized with formalin-inactivated RS virus (ref. 31). Furthermore,evaluation of RS virus formalin-inactivated vaccine in cotton rats alsoshowed that upon live virus challenge, immunized animals developedenhanced pulmonary histopathology (ref. 32).

The mechanism of disease potentiation caused by formalin-inactivated RSvirus vaccine preparations remains to be defined but is a major obstaclein the development of an effective RS virus vaccine. The potentiationmay be partly due to the action of formalin on the F and Gglycoproteins. Additionally, a non-RS virus specific mechanism ofdisease potentiation has been suggested, in which an immunologicalresponse to contaminating cellular or serum components present in thevaccine preparation could contribute, in part, to the exacerbateddisease (ref. 33). Indeed, mice and cotton rats vaccinated with a lysateof HEp-2 cells and challenged with RS virus grown on HEp-2 cellsdeveloped a heightened pulmonary inflammatory response.

Furthermore, RS virus glycoproteins purified by immunoaffinitychromatography using elution at acid pH were immunogenic and protectivebut also induced immunopotentiation in cotton rats (refs. 31, 34).

Influenza virus infection is one of the most common causes ofrespiratory tract diseases. Typically, the disease results in a highfever, usually 100° F. to 103° F. in adults, often higher in children,and respiratory symptoms, such as sore throat, running or stuffy nose,as well as headache, muscle aches and extreme fatigue. In a typicalyear, influenza is associated with about 20,000 deaths in the US, andmany more hospitalizations (CDC).

Influenza viruses are divided into three types, designated A, B and C.Types A and B are responsible for epidemics that occur almost everywinter. Influenza viruses continually change over time by mutation,which is termed antigenic drift.

Influenza A viruses are classified into sub-types on the basis of twosurface antigens, hemagglutinin (H) and neuraminidase (N). Threesubtypes of the hemagglutinin (H1, H2, H3) and two sub-types ofneuraminidase (N1, N2) are recognized among influenza A viruses thathave caused widespread human diseases. Immunity to these antigensreduces the likelihood of infections and lessens the severity of thedisease if infection occurs.

As a result of antigenic drift, major epidemics of respiratory diseasecaused by new variants of influenza continue to occur. Thus, theantigenic characteristics of the circulating strains provide the basisfor selecting the virus strains included in each year's vaccine.

Although there are many actual and potential benefits of vaccines thatcombine antigens to confer protection against multiple pathogens, thesecombinations may have a detrimental effect on the immunogenicity of theindividual components.

As described above, RSV and influenza virus infections are prevalent inthe adult population and particularly the elderly and it would bedesirable to confer protection against such infection by theadministration of a single vaccine composition. However, any potentialdetrimental effect of combining immunogens suitable for conferringprotection against both RSV and influenza virus in a single formulationare unknown.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that combining a mixture of RSVproteins with non-virulent influenza virus in a vaccine formulationprovides an immune response which is substantially the same as theresponse obtained by administration of the components individually.Accordingly, there is no observed detrimental effect on theimmunogenicity of the individual components by combining them in asingle formulation. The inventors have also surprisingly found that, inthe presence of the non-virulent influenza virus, an enhanced immuneresponse to the mixture of RSV proteins can be obtained by formulatingthe immunogenic composition with an adjuvant.

Accordingly, in one aspect of the present invention, there is provided amultivalent immunogenic composition for conferring protection in a hostagainst disease caused by infection by respiratory syncytial virus (RSV)and influenza virus, which comprises (a) an immunoeffective amount of amixture of purified fusion (F) protein, attachment (G) protein andmatrix (M) protein of RSV, and (b) an immunoeffective amount of anon-virulent influenza virus preparation. The immunogenic compositionpreferably is formulated as a vaccine for in vivo administration to thehost, particularly an adult human host (at least 18 years of age),wherein the individual components (a) and (b) of the composition areformulated such that the immunogenicity of the individual components (a)and (b) is not impaired.

The immunogenic compositions of the invention may be formulated asmicroparticles, capsules, ISCOMs or liposomes. The immunogeniccompositions may further comprise at least one other immunogenic orimmunostimulating material, which may be at least one adjuvant or atleast one immunomodulator, such as cytokines including IL-2.

The immunogenic composition provided herein may further comprise anadjuvant, particularly an adjuvant which imparts an enhanced immuneresponse to RSV when compared to the RSV mixture formulated with theadjuvant in the absence of the non-virulent influenza virus preparation.

The at least one adjuvant may be selected from the group consisting ofaluminum phosphate, aluminum hydroxide, QS21, Quil A or derivatives orcomponents thereof, calcium phosphate, calcium hydroxide, zinchydroxide, a glycolipid analog, an octodecyl ester of an amino acid, amuramyl dipeptide, polyphosphazene, ISCOPREP, a lipoprotein, ISCOMmatrix, DC-Chol, DDBA, and other adjuvants and bacterial toxins,components and derivatives thereof as, for example, described in U.S.Ser. No. 08/258,228 filed Jun. 10, 1994, assigned to the assignee hereofand the disclosure of which is incorporated herein by reference thereto(WO 95/34323). Under particular circumstances, adjuvants that induce aTh1 response are desirable. Advantageous combinations of adjuvants aredescribed in copending U.S. patent application Ser. No. 08/483,856 filedJun. 7, 1995, assigned to the Assignee hereof and the disclosure ofwhich is incorporated herein by reference (WO 95/34308).

Preferably, the adjuvant in the polyphosphazene, i.e.poly-di(carboxylatophenoxy)-phosphazene (PCPP).

The immunogenic composition of the invention may be formulated in singledosage form, wherein the mixture of RSV proteins is present in an amountof about 10 to about 200 μg, preferably about 50 to about 100 μg, andthe non-virulent influenza virus preparation is present in an amount ofabout 1 to about 100 μg, preferably about 10 to about 75 μg.

The fusion (F) protein may comprise multimeric fusion (F) proteins,which may include, when analyzed under non-reducing conditions,heterodimers of molecular weight approximately 70 kDa and dimeric andtrimeric forms.

The attachment (G) protein may comprise, when analyzed undernon-reducing conditions, oligomeric G protein, G protein of molecularweight approximately 95 kDa and G protein of molecular weightapproximately 55 kDa.

The matrix (M) protein may comprise, when analyzed under non-reducingconditions, protein of molecular weight approximately 28 to 34 kDa.

The RSV protein mixture employed herein, when analyzed by reducedSDS-PAGE analysis, may comprise the fusion (F) protein comprising an F₁subunit of molecular weight approximately 48 kDa and an F₂ subunit ofabout 23 kDa, the attachment (G) protein comprising a G protein ofmolecular weight approximately 95 kDa and a G protein of molecularweight approximately 55 kDa, and the matrix (M) protein comprising an Mprotein of approximately 31 kDa.

The RSV protein mixture employed in the invention may comprise the F, Gand M proteins in the relative proportions of:

-   -   F about 35 to about 70 wt %    -   G about 5 to about 30 wt %    -   M about 10 to about 50 wt %        When analyzed by SDS-PAGE under reducing conditions and        densitometric scanning following silver staining, the ratio of        F₁ subunit of molecular weight approximately 48 kDa to F₂        subunit of molecular weight approximately 23 kDa in is this        mixture may be approximately between 1:1 and 2:1. The mixture of        F, G and M proteins may have a purity of at least about 75%,        preferably at least about 85%.

The mixture employed herein in accordance with this aspect of theinvention, having regard to the method of isolation employed herein asdescribed below, is devoid of monoclonal antibodies and devoid of lentillectin and concanavalin A.

The RSV proteins provided in the mixture of proteins employed hereingenerally are substantially non-denatured by the mild conditions ofpreparation and may comprise RSV proteins from one or both of subtypesRSV A and RSV B.

The composition and manner of preparation of the mixture of RSV proteinsis fully described in U.S. patent application Ser. No. 08/679,060, filedJul. 12, 1996, and in published PCT Application WO 98/02457, thedisclosures of which are incorporated herein by reference. As describedtherein, the mixture of RSV proteins may be obtained by coisolating andcopurifying the mixture from the virus. RSV cells are grown in a cellculture and separated from the cell culture. The F, G and M proteins aresolubilized from the separated virus and the solubilized RSV protein arecoisolated and copurified. Such coisolation and copurification may beeffected by loading the solubilized proteins onto an ion-exchangematrix, preferably a calcium phosphate matrix, specifically ahydroxyapatite matrix, and selectively eluting the F, G and M proteinsfrom the ion-exchange matrix. The grown virus may first be washed withurea to remove contaminants without substantially removing F, G and Mproteins.

The non-virulent influenza preparation employed herein usually comprisesa plurality of different non-virulent influenza virus strains, includingattenuated viruses, which may be cold adapted. Conventionally influenzavirus vaccines are formulated annually based on the strains prevalentand extant during the provisions flu season and may comprise two, threeor more different strains. Such influenza virus preparation may berendered non-virulent in any convenient manner, such as by inactivationwith any convenient inactivating agent, such as formaldehyde. Thenon-virulent influenza preparation may comprise influenza antigens, suchas HA, NA, NP, M, PBI, NS1, NS2 or PB2, which may be isolated fromattenuated or inactivated virus or may be prepared recombinantly.

In a further aspect of the present invention, there is provided a methodof immunizing a human host against disease caused by infection byrespiratory syncytial virus (RSV) and influenza virus, which comprisesadministering to the host an immunoeffective amount of the immunogeniccomposition provided herein.

The immunogenic composition preferably is formulated as a vaccine for invivo administration to the host wherein the individual components (a)and (b) of the composition are formulated such that the immunogenicityof the individual components (a) and (b) is not impaired. Theformulation provided herein enables the elderly to be protected by suchimmunization.

The present invention provides, in an additional aspect thereof, amethod of producing a vaccine for protection against disease caused byrespiratory syncytial virus (RSV) infection and by influenza virusinfection, comprising administering the immunogenic composition providedherein to a test host to determine the amount of and frequency ofadministration thereof to confer protection against disease caused byRSV and by influenza virus; and formulating the immunogenic compositionin a form suitable for administration to a treated host in accordancewith the determined amount and frequency of administration. The treatedhost may be a human.

The present invention further extends to the immunogenic composition ofthe invention when used as a vaccine. In addition, the present inventionincludes the use of (a) a mixture of purified fusion (F) protein,attachment (G) protein and matrix (M) protein of RSV and (b) anon-virulent influenza virus preparation in the manufacture of a vaccinefor conferring protection in a host against disease caused by RSV and byinfluenza virus.

Advantages of the present invention include the provision of a singlevaccine formulation which permits immunization of the elderly againstdisease caused by infection by RSV and influenza virus in a singleimmunization regimen.

BRIEF DESCRIPTION OF DRAWINGS

In each of the Figures, a common legend is used for identification ofthe immunogens used in the experiments for which the data is presentedin the Figures is as follows:

(a) phosphate buffered saline (PBS); (b) 200 μg PCPP adjuvant; (c)1.5×10⁶ pfu live RSV; (d) 200 to 400 HA units live influenza; (e) 5 μgFluzone® vaccine with PCPP adjuvant; (f) 1 μg RSV vaccine with PCPPadjuvant; (g) 5 μg Fluzone® vaccine plus 1 μg RSV vaccine with PCPPadjuvant; (h) 5 μg Fluzone® vaccine plus 1 μg RSV vaccine with noadjuvant; (i) 5 μg Fluzone® vaccine with no adjuvant; (j) 1 μg RSVvaccine with no adjuvant.

FIG. 1 shows the anti-RSV F immunoglobulin titres in mice immunized witheach of the immunogens;

FIG. 2 shows the RSV plaque reduction titres in mice immunized with eachof the immunogens;

FIG. 3 shows the RSV titres in the lungs of mice immunized with each ofthe immunogens and then challenged with live RSV;

FIG. 4 shows the anti-A/Johannesburg influenza immunoglobulin titres inmice immunized with each of the immunogens;

FIG. 5 shows the anti-A/Texas influenza immunoglobulin titres in miceimmunized with each of the immunogens;

FIG. 6 shows the anti-B/Harbin influenza immunoglobulin titres in miceimmunized with each of the immunogens;

FIG. 7 shows the anti-A/Johannesburg influenza hemagglutinationinhibition titres in mice immunized with each of the immunogens;

FIG. 8 shows the anti-A/Texas influenza hemagglutination inhibitiontitres in mice immunized with each of the immunogens; and

FIG. 9 shows the anti-B/Harbin influenza hemagglutination inhibitiontitres in mice immunized with each of the immunogens.

GENERAL DESCRIPTION OF INVENTION

The mixture of F, G and M proteins of RSV used herein may be coisolatedand copurified from RS virus. As described in the aforesaid U.S. Ser.No. 08/679,060 and WO 98/02457, the virus is grown on a vaccine qualitycell line, such as VERO cells and human diploid cells, such as MRC5 andWI38, and the grown virus is harvested. The fermentation may be effectedin the presence of fetal bovine serum (FBS) and trypsin.

The viral harvest is filtered and then concentrated, typically usingtangential flow ultrafiltration with a membrane of desired molecularweight cut-off, and diafiltered. The virus harvest concentrate may becentrifuged and the supernatant discarded. The pellet followingcentrifugation may first be washed with a buffer containing urea toremove soluble contaminants while leaving the F, G and M proteinssubstantially unaffected, and then recentrifuged. The pellet from thecentrifugation then is detergent extracted to solubilize the F, G and Mproteins from the pellet. Such detergent extraction may be effected byresuspending the pellet to the original harvest concentrate volume in anextraction buffer containing a detergent, such as a non-ionic detergent,including TRITON® X-100, a non-ionic detergent which is octadienylphenol (ethylene glycol)₁₀. Other detergents which may be used includeoctylglucoside and Mega detergents.

Following centrifugation to remove non-soluble proteins, the F, G and Mprotein extract is purified by chromatographic procedures. The extractmay first be applied to an ion exchange chromatography matrix to permitbinding of the F, G and M proteins to the matrix while impurities arepermitted to flow through the column. The ion-exchange chromatographymatrix may be any desired chromatography material, particularly acalcium phosphate matrix, specifically hydroxyapatite, although othermaterials, such as DEAE and TMAE and others, may be used.

The bound F, G and M proteins then are coeluted from the column by asuitable eluant. The resulting copurified F, G and M proteins may befurther processed to increase the purity thereof.

The purified F, G and M proteins employed herein may be in the form ofhomo and hetero oligomers including F:G heterodimers and includingdimers, tetramers and higher species. The RSV protein preparationsprepared following this procedure demonstrated no evidence of anyadventitious agent, hemadsorbing agent or live virus.

The influenza virus vaccine utilized herein is a sterile suspensionprepared from influenza virus propagated in chicken embryos. Thevirus-containing allantoic fluids are harvested and inactivated withformaldehyde. The virus is then concentrated and purified in a linearsucrose density gradient solution, using a continuous flow centrifuge.The virus is then chemically disrupted using Triton® X-100 producing asplit-antigen. The split-antigen is then further purified by chemicalmeans and suspended in sodium phosphate-buffered isotonic sodiumchloride solution. Gelatin (0.05%) is then added as a stabilizer andthimerosol (1:10,000) is added as a preservative.

The commercial split flu antigen vaccine (Fluzone®) as used herein andprepared following the above procedure was obtained from ConnaughtLaboratories Inc., Swiftwater, Pa., USA.

As set forth in detail in the Examples below, various combinations ofRSV-A subunit vaccine and trivalent influenza vaccine were prepared withor without PCPP adjuvant and were tested for their immunogenicity incomparison to several controls. In the immunization studies, details ofwhich are provided below, Balb/c mice were immunized with two injectionsof immunogen given three weeks apart. Bleeds were collected to monitorthe immune response and, at the end of the study, the mice werechallenged with either influenza or RSV to determine whether protectionwas obtained.

Details of the results obtained are set forth in the Examples below andin FIGS. 1 to 9. It was found that neither the RSV nor influenza antigeninterfered or impaired the immunogenicity of the other, both inadjuvanted and unadjuvanted form. In addition, when adjuvanted, thecombination of the RSV and influenza immunogen produced an enhancedimmune response to RSV in comparison to the absence of the influenzaimmunogens.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis and treatment of respiratory syncytialvirus and influenza virus infections, and the generation ofimmunological agents. A further non-limiting discussion of such issue isfurther presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from mixtures comprising immunogenic F, G and M proteins of RSValong with a non-virulent influenza virus preparation. The immunogeniccomposition elicits an immune response which produces antibodies,including anti-RSV antibodies including anti-F, anti-G and anti-Mantibodies as well as anti-influenza antibodies to each of the strainspresent in the formulation. Such antibodies may be viral neutralizingand/or anti-fusion antibodies.

Immunogenic compositions including vaccines may be prepared asinjectables, as liquid solutions, suspensions or emulsions. The activeimmunogenic ingredients may be mixed with pharmaceutically acceptableexcipients which are compatible therewith. Such excipients may includewater, saline, dextrose, glycerol, ethanol, and combinations thereof.The immunogenic compositions and vaccines may further contain auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,or adjuvants to enhance the effectiveness thereof. Immunogeniccompositions and vaccines may be administered parenterally, bysubcutaneous, intradermal or intramuscular injection. Alternatively, theimmunogenic compositions formulated according to the present invention,may be formulated and delivered in a manner to evoke an immune responseat mucosal surfaces. Thus, the immunogenic composition may beadministered to mucosal surfaces by, for example, the nasal or oral(intragastric) routes. Alternatively, other modes of administrationincluding suppositories and oral formulations may be desirable. Forsuppositories, binders and carriers may include, for example,polyalkalene glycols or triglycerides. Such suppositories may be formedfrom mixtures containing the active immunogenic ingredient(s) in therange of about 0.5 to about 10%, preferably about 1 to 2%. Oralformulations may include normally employed carriers such as,pharmaceutical grades of saccharine, cellulose and magnesium carbonate.These compositions can take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders and containabout 1 to 95% of the active ingredients, preferably about 20 to about75%.

The immunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, immunogenic and protective. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizeantibodies, and, if needed, to produce a cell-mediated immune response.Precise amounts of active ingredients required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges arereadily determinable by one skilled in the art and may be of the orderof micrograms to milligrams of the active ingredient(s) per vaccination.Suitable regimes for initial administration and booster doses are alsovariable, but may include an initial administration followed bysubsequent booster administrations. The dosage may also depend on theroute of administration and will vary according to the size of the host.

The concentration of the active ingredients in an immunogeniccomposition according to the invention is in general about 1 to 95%. Avaccine which contains antigenic material of only one pathogen is amonovalent vaccine.

Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants. Adjuvants enhance the immunogenicity ofan antigen but are not necessarily immunogenic themselves. Adjuvants mayact by retaining the antigen locally near the site of administration toproduce a depot effect facilitating a slow, sustained release of antigento cells of the immune system. Adjuvants can also attract cells of theimmune system to an antigen depot and stimulate such cells to elicitimmune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccharides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are formulated to enhance the host immuneresponses. Thus, adjuvants have been identified that enhance the immuneresponse to antigens delivered parenterally. Some of these adjuvants aretoxic, however, and can cause undesirable side-effects, making themunsuitable for use in humans and many animals. Indeed, only aluminumhydroxide and aluminum phosphate (collectively commonly referred to asalum) are routinely used as adjuvants in human and veterinary vaccines.The efficacy of alum in increasing antibody responses to diphtheria andtetanus toxoids is well established. While the usefulness of alum iswell established for some applications, it has limitations. For example,alum is ineffective for influenza vaccination and usually does notelicit a cell mediated immune response. The antibodies elicited byalum-adjuvanted antigens are mainly of the IgG1 isotype in the mouse,which may not be optimal for protection by some vaccinal agents.

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria in mineral oil, Freund's incomplete adjuvant,bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

To efficiently induce humoral immune responses (HIR) and cell-mediatedimmunity (CMI), immunogens are often emulsified in adjuvants. Manyadjuvants are toxic, inducing granulomas, acute and chronicinflammations (Freund's complete adjuvant, FCA), cytolysis (saponins andPluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPSand MDP). Although FCA is an excellent adjuvant and widely used inresearch, it is not licensed for use in human or veterinary vaccinesbecause of its toxicity.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

Methods of determining tissue culture infectious dose₅₀ (TCID₅₀/mL),plaque and neutralization titres, not explicitly described in thisdisclosure are amply reported in the scientific literature and wellwithin the scope of those skilled in the art. Protein concentrationswere determined by the bicinchoninic acid (BCA) method as described inthe Pierce Manual (23220, 23225; Pierce Chemical company, U.S.A.),incorporated herein by reference.

CMRL 1969 and Iscove's Modified Dulbecco's Medium (IMDM) culture mediawere used for cell culture and virus growth. The cells used in thisstudy are vaccine quality African green monkey kidney cells (VERO lotM6) obtained from Institut Mérieux. The RS viruses used were the RSvirus subtype A (Long and A2 strains) obtained from the American Typeculture Collection (ATCC), a recent subtype A clinical isolate and RSVsubtype B clinical isolate from Baylor College of Medicine.

Example 1

This Example illustrates the production of RSV on a mammalian cell lineon microcarrier beads in a 150 L controlled fermenter.

Vaccine quality African green monkey kidney cells (VERO) at aconcentration of 10⁵ cells/mL were added to 60 L of CMRL 1969 medium, pH7.2 in a 150 L bioreactor containing 360 g of Cytodex-1 microcarrierbeads and stirred for 2 hours. An additional 60 L of CMRL 1969 was addedto give a total volume of 120 L. Fetal bovine serum was added to achievea final concentration of 3.5%. Glucose was added to a finalconcentration of 3 g/L and L-glutamine was added to a finalconcentration of 0.6 g/L. Dissolved oxygen (40%), pH (7.2), agitation(36 rpm) and temperature (37° C.) were controlled. Cell growth, glucose,lactate and glutamine levels were monitored. At day 4, the culturemedium was drained from the fermenter and 100 L of E199 media (no fetalbovine serum) was added and stirred for 10 minutes. The fermentor wasdrained and filled again with 120 L of E199 media.

An RSV inoculum of RSV subtype A was added at a multiplicity ofinfection (M.O.I.) of 0.001 and the culture was then maintained for 3days before one-third to one-half of the medium was drained and replacedwith fresh medium. On day 6 post-infection, the stirring was stopped andthe beads allowed to settle. The viral culture fluid was drained andfiltered through a 20 mm filter followed by a 3 mm filter prior tofurther processing.

The clarified viral harvest was concentrated 75- to 150-fold usingtangential flow ultrafiltration with 300 NMWL membranes and diafilteredwith phosphate buffered saline containing 10% glycerol. The viralconcentrate was stored frozen at −70° C. prior to further purification.

Example 2

This Example illustrates the process of purifying RSV subunits from aviral concentrate of RSV subtype A.

A solution of 50% polyethylene glycol-8000 was added to an aliquot ofvirus concentrate prepared as described in Example 1 to give a finalconcentration of 6%. After stirring at room temperature for one hour,the mixture was centrifuged at 15,000 RPM for 30 min in a Sorvall SS-34rotor at 4° C. The viral pellet was suspended in 1 mM sodium phosphate,pH 6.8, 2 M urea, 0.15 M NaCl, stirred for 1 hour at room temperature,and then recentrifuged at 15,000 RPM for 30 min. in a Sorvall SS-34rotor at 4° C. The viral pellet was then suspended in 1 mM sodiumphosphate, pH 6.8, 50 mM NaCl, 1% Triton X-100 and stirred for 30minutes at room temperature. The insoluble virus core was removed bycentrifugation at 15,000 RPM for 30 min. in a Sorval SS-34 rotor at 4°C. The soluble protein supernatant was applied to a column of ceramichydroxyapatite (type II, Bio-Rad Laboratories) and the column was thenwashed with five column volumes of 1 mM sodium phosphate, pH 6.8, 50 mMNaCl, 0.02% Triton X-100. The RSV subunit composition from RSV subtypeA, containing the F, G and M proteins, was obtained by eluting thecolumn with 10 column volumes of 1 mM sodium phosphate, pH 6.8, 400 mMNaCl, 0.02% Triton X-100.

Example 3

This Example illustrates the production of influenza virus.

The influenza virus vaccine utilized herein is a sterile suspensionprepared from influenza virus propagated in chicken embryos. The viruscontaining allantoic fluids are harvested and inactivated withformaldehyde. The virus is then concentrated and purified in a linearsucrose density gradient solution, using a continuous flow centrifuge.The virus is then chemically disrupted using Triton® X-100 producing asplit-antigen. The split-antigen is then further purified by chemicalmeans and suspended in sodium phosphate-buffered isotonic sodiumchloride solution. Gelatin (0.05%) is then added as a stabilizer andthimerosol (1:10,000) is added as a preservative.

The commercial vaccine (Fluzone®) as used herein, prepared as describedabove was obtained from Connaught Laboratories Inc., Swiftwater, Pa.,USA.

Example 4

This Example illustrates the immunization protocol used in the micestudies.

Mice were bled one day prior to the first immunization and also on days22 and 28 of the study. Immunizations were done on days 1 and 22. Bothimmunizations were administered intramuscularly in the thigh muscle.Each immunization was done at two injection sites (both right and leftthigh muscles; 0.05 ml/site). The dose of RSV vaccine was 1 μg totalprotein and the dose of Fluzone® (split flu antigen) vaccine was 5 μgtotal protein per dose. The RSV or Fluzone® (split flu antigen) vaccineswere administered in the presence or absence of adjuvant. The adjuvantused was poly-di(carboxylatophenoxy)-phosphazene (PCPP) given at 200μg/dose. Mice that received live RSV (A2 strain) as the immunogen weregiven 1.5×10⁶ pfu/dose intranasally. Mice that received live influenzavirus (A/Taiwan Strain) as the immunogen were given 200 to 400 HAU/doseintraperitonally. Virus challenge with either RSV or influenza wasadministered intranasally on day 29 using the same dose as given for thelive virus immunized mice. All animals were sacrificed on day 33. Lungswere removed and frozen immediately in liquid nitrogen for laterdetermination of virus titre.

Example 5

This Example illustrates the determination of RSV titres in the lungs ofmice.

Mouse lungs were removed at the time of sacrifice, quick frozen inliquid nitrogen, and then stored at −70° C. until assayed for virustitre. To process the lungs, they were thawed, weighed and thenhomogenized in Dulbecco's Modified Eagles (DME) tissue culture mediacontaining 10% fetal bovine serum. The homogenate was centrifuged at200×g for 15 min to remove cell debris and the supernatant wascollected. The supernatant was assayed for RSV titres using the RSVplaque assay, as described in Example 6.

When the mice were challenged with RSV, FIG. 3, again a good immuneresponse was observed in the combination and adjuvant (lane g) showingvery low titres in the lungs, comparable to the RSV alone (lane f) orthe live RSV (lane c). This also shows the lack of interference betweenthe influenza component and the RSV components.

Example 6

This Example describes the RSV plaque assay.

Vero cells were grown in CMRL 1969 media plus 10% FBS for RSVtitrations. Test samples were diluted serially in 10-fold steps andadded to 24-well plates containing confluent Vero cells for 1 to 2hours. Following adsorption, the sample was removed and replaced withmedia containing 0.75% methyl cellulose. After 4 to 5 days incubation,virus plaques were detected by probing the wells with monoclonal anti-Fantibody. Bound antibody was visualized using sequential incubation withhorse radish peroxidase-conjugated donkey anti-mouse immunoglobulin and4-choro-1-napthol/H₂O₂. Plaques were scored manually.

Example 7

This Example describes the RSV plaque reduction assay.

Test sera were heat-inactivated at 56° C. for 30 minutes. Samples werediluted in four-fold serial steps and mixed with an equal volume of RSVA (long strain; 30–70 PFU) in assay media containing 10% guinea pigcomplement. After one hour incubation at 37° C. the mixture wasinoculated onto Vero cells for 1 to 2 hours. This was followed by anoverlay with 0.75% methyl cellulose and incubation for 4 to 5 days.Virus plaques were detected as described for the RSV plaque assay inExample 6. The neutralization titre is expressed as the reciprocal ofthe dilution which results in 60% reduction in plaque formation (asdetermined by linear interpolation analysis).

The enhancement of the RSV response is illustrated in FIG. 2 whereplaque reduction titres were looked at. The RSV/Flu combination (lane g)again shows a higher titre than the RSV alone (lane f).

Example 8

This Example describes the mouse anti-RSV F antibody ELISA.

Anti-F immunoglobulin antibody titres in mouse sera were measured in anantigen-specific ELISA employing native F protein as the solid-phasecoat. The F protein was purified by immunoaffinity chromatography usingan immobilized anti-F monoclonal antibody. Wells were coated with Fprotein, then blocked with 1% BSA in PBS. Dilutions of test serumsamples were added, and after incubation, the wells were washed againwith 1% BSA. The bound F-specific antibodies were detected with horseradish peroxidase-labeled antibody specific for mouse IgG (H+L chains),followed after further washing by tetramethylbenzidine plus hydrogenperoxide substrate. Colour formation was measured at 450 nm in anautomatic plate reader. The antibody titre is expressed as thereciprocal of the greatest four-fold dilution at which optical densityremains double that of a negative control.

As can be seen from FIG. 1, RSV-F IgG antibody response was observed inthe RSV/Flu immunizations (lanes g+h), either with or without adjuvant.These results are comparable to RSV immunization alone (lane f) and infact the combination immunization (lane g) shows an enhanced RSVresponse over RSV alone (lane f). This shows that there was nointerference between the influenza component and the RSV components.

Example 9

This Example describes the mouse anti-influenza antibody ELISA.

Influenza strain-specific antibody titres in mouse sera were measuredusing microitre plates coated with the appropriate influenza strain(A/Texas, A/Johannesburg, or B/Harbin). Plate processing and developmentwas done as described for the RSV-F antibody ELISA in Example 8.

As can be seen from FIGS. 4, 5, 6, all three strains of influenzaeelicited a good antibody response to influenza virus in the combinationRSV/Flu administration (lane g). This was comparable to the flu vaccineadministered alone (lane e). This again shows that the combinationvaccine did not reduce or interfere with the immune response to theinfluenza component.

Example 10

This Example describes the influenza hemagglutination inhibition assay.

Sera samples were heated at 56° C. for 30 minutes to inactivatecomplement 1 and then treated with trypsin and potassium periodate todestroy endogenous inhibitors of hemagglutination. Serially dilutedantisera were tested for their ability is to inhibit the agglutinationof chicken red blood cells by four HA units of influenza virus (A/Texas,A/Johannesburg, or B/Harbin) in a standard hemagglutination inhibitionassay.

FIGS. 7, 8 and 9 shows that the combination vaccine (lane g) produced asgood haemagglutinin (HA) titres as the Flu vaccine as its own. Againthis illustrates that the RSV component did not interfere with theeliciting of a good influenza immune response, in this case as measuredby HAI.

SUMMARY OF THE DISCLOSURE

In summary of the disclosure, the present invention provides amultivalent immunogenic composition comprising an RSV protein subunitcomponent and a non-virulent influenza virus preparation wherein theactive ingredients do not interfere with the immunogenicity of the otherand which is suitable for administration to adults and the elderly.Modifications are possible with the scope of the invention.

REFERENCES

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1. A multi-valent immunogenic composition, which comprises: (a) amixture of purified fusion (F), attachment (G) and matrix (M) protein ofRSV, (b) a non-virulent influenza virus preparation, and (c) anadjuvant, said immunogenic composition being formulated for in vivoadministration to the host wherein the individual components (a) and (b)of the composition are formulated such that the immunogenicity of theindividual components (a) and (b) is not impaired, wherein said adjuvantis poly-di(carboxylatophenoxy)-phosphazene (PCPP) and is present in anamount which imparts an enhanced immune response to RSV when compared tothe mixture (a) formulated with the adjuvant in the absence of thenon-virulent virus preparation, and wherein said non-virulent influenzavirus preparation is prepared as follows: (i) preparing a sterilesuspension of influenza virus propagated in chicken embryos; (ii)inactivating the virus in the suspension with formaldehyde; (iii)concentrating and purifying the virus in a linear sucrose densitygradient solution, where said concentrating and purifying is performedusing a continuous flow centrifuge; (iv) chemically disrupting thepurified inactivated virus using Triton X-100, resulting in asplit-antigen; (v) further purifying the split-antigen by chemicalmeans; (vi) suspending the purified split-antigen in a sodiumphosphate-buffered isotonic sodium chloride solution; and (vii) addingto the split-antigen solution a 0.05% Gelatin stabilizer and thepreservative thimerosol at a 1:10,000 concentration.
 2. The immunogeniccomposition of claim 1 wherein said mixture (a) is present in an amountof about 10 to about 200 μg and (b) is present in an amount of about 1to about 100 μg, in a single dose.
 3. The immunogenic composition ofclaim 1 wherein said fusion (F) protein comprises multimeric fusion (F)proteins.
 4. The immunogenic composition of claim 3 wherein, whenanalyzed under non-reducing conditions, said multimeric fusion (F)protein includes heterodimers of molecular weight approximately 70 kDaand dimeric and trimeric forms.
 5. The immunogenic composition of claim1 wherein, when analyzed under non-reducing conditions, said attachment(G) protein comprises G protein of molecular weight approximately 95 kDaand G protein of molecular weight approximately 55 kDa and oligomeric Gprotein.
 6. The immunogenic composition of claim 1 wherein, whenanalyzed by SDS-PAGE under non-reducing conditions, said matrix (M)protein comprises M protein of molecular weight approximately 28 to 34kDa.
 7. The immunogenic composition of claim 1 wherein, when analyzed byreduced SDS-PAGE analysis, said fusion (F) protein comprises an F₁subunit of molecular weight approximately 48 kDa and an F₂ subunit ofmolecular weight approximately 23 kDa, said attachment (G) proteincomprises a G protein of molecular weight approximately 95 kDa and a Gprotein of molecular weight approximately 55 kDa, and said matrix (M)protein comprises an M protein of approximately 31 kDa.
 8. Theimmunogenic composition of claim 1 wherein said F, G and M proteins arepresent in mixture (a) in the relative proportions of: F from about 35to about 70 wt % G from about 5 to about 30 wt % M from about 10 toabout 50 wt %.
 9. The immunogenic composition of claim 8 wherein, whenanalyzed by SDS-PAGE under reducing conditions and silver stained, theratio of F₁ subunit of molecular weight approximately 48 kDa to F₂subunit of molecular weight approximately 23 kDa is between 1:1 to about2:1 as determined by scanning densitometry.
 10. The immunogeniccomposition of claim 9 wherein said mixture is at least about 75% pure.11. The immunogenic composition of claim 1 wherein said RSV proteins insaid mixture are from one or both of subtypes RSV A and RSV B.
 12. Amethod of inducing an immune response in a human host, which comprisesadministering to the host the immunogenic composition of claim
 1. 13.The method of claim 12 wherein said host is a human host of at least 18years of age.